Project Summary/Abstract Approximately 50,000 new diagnoses of Parkinson?s disease (PD) occur each year, which amounts to half a million people currently living with the disease in the USA. This disease burden will only increase as the population continues to age. Current treatments only reduce disease symptoms but do not treat the underlying cause of neurodegeneration. The long-term goal of our research is to identify molecular targets associated with PD pathogenesis and develop specific therapies which abrogate neuronal death and halt progression of the disease. The causes of PD are varied, both genetic and environmental. A major hypothesis in the field supports an interaction between genetic susceptibilities and environmental factors (termed GxE), such as pesticide/mitochondrial toxin exposure, which can create oxidative/nitrosative stress. To this end, many studies have implicated reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis and progression of neurodegenerative diseases including PD. Cysteine thiol oxidation by ROS/RNS is of particular interest because ROS and RNS have been shown to oxidize the same cysteines in several proteins to form sulfenic (-SOH), sulfinic (-SO2H), or sulfonic (-SO3H) acid and nitrosothiol (-SNO) posttranslational modifications, respectively. Our lab has shown that these modifications on specific proteins can elicit different cellular responses and can mimic rare genetic mutations of the gene encoding the protein (e.g., for Parkin, PINK1 and others). Research on global cellular effects from changes in the stoichiometries between these oxidative thiol modifications is scarce. This is likely due to the technical challenges associated with the high reactivity of thiols and the short half-lives of oxidative modifications. This thiol redox ratio is important to assess if thiol modifications are contributing to a disease phenotype or if they are nonspecific consequences of increased/aberrant ROS/RNS production (i.e., inhibition requires higher modification levels compared to an activation). Recent advances in molecular probes specific to cysteine sulfenylation and nitrosylation provide new technologies that allow for quantitative analyses of these oxidized species as well as relate respective modifications to cellular disease phenotypes. The PI has extensive experience utilizing thiol probes in chemoproteomic analyses. A novel detection strategy will be developed to monitor differential oxidation of cysteine residues (by ROS/RNS) in a quantitative fashion. This will be employed in an isogenic human induced pluripotent stem cell (iPSC) neuronal model system of PD to test the central hypothesis: Cysteine S- nitrosylation and S-sulfenylation can occur sequentially and differentially on reactive thiols of specific proteins, contributing to neurodegeneration. Testing this hypothesis strengthens growing evidence that these oxidized thiol moieties both contribute to disease progression and differentially affect specific protein targets. Novel protein targets of ROS/RNS will be characterized and validated with in vivo brain proteomic analysis using tissue acquired from human PD patients and controls.